Hemostatic foam

ABSTRACT

The invention discloses a pharmaceutical hemostatic liquid foam base preparation comprising albumin as foaming agent and a fibrinogen precipitating substance and optionally a coagulation inducing agent, wherein albumin as foaming agent is present in native form; a method for the production of a transient hemostatic liquid foam; the transient hemostatic liquid foam; and a kit for making the foam.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 U.S. National Stage Entry ofInternational Application Serial No. PCT/EP2013/075852 (WO 2014/086996)filed Dec. 6, 2013, which claims the benefit of priority to U.S.Provisional Patent Application No. 61/734,544 filed Dec. 7, 2012. Thisapplication is also related to U.S. patent application Ser. No.14/099,712 filed Dec. 6, 2013. The entire content of each of the abovefilings is incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical hemostatic foamformulations.

BACKGROUND OF THE INVENTION

Foams are generally understood to represent materials in which a gas,such as air, is finely dispersed in a liquid in a form that is stableover some time. The liquid is found in the form of thin films betweenthe bubbles. The stabilizing element of such gas dispersions is thesurface tension of the liquid phase. The surface tension can beinfluenced by the presence of surface-active agents in the fluid phase,which help to stabilize the films against collapse. These foams aregenerally referred to as closed pore foams, as there is no connectionbetween the gas spaces of each individual bubble. If the liquid filmsare stabilized by other means, e.g. by chemical reactions that transformthe liquid phase into a viscoelastic solid, then the formation of openpore foams is possible, in which the fluid bridges are removed in acontrolled manner, e.g. by evaporation or other processes. Thetransformation of the liquid films into a viscoelastic solid materialalso serves to effectively stabilize the foams against rapid collapse.

It is an object of the present invention to provide improved hemostaticfoams which can be safely injected into tissue voids with low visibilityand which are effective with no need for approximation. In addition, itis important that such a foam hemostat does not present a risk ofobstruction or compression of pressure sensitive organs or tissues dueto excessive swelling. Such a foam should be specifically applicable inneuro/spine, laparoscopic and cardiovascular surgery.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a pharmaceutical hemostaticliquid foam base preparation comprising albumin as foaming agent and afibrinogen precipitating substance and optionally a coagulation inducingagent, wherein albumin as foaming agent is present in native form.

Another aspect of the present invention is a method for preparing apharmaceutical hemostatic liquid foam, wherein a pharmaceuticalhemostatic liquid foam base preparation according to the presentinvention is contacted with a foaming gas so as to obtain apharmaceutical hemostatic liquid foam.

Another aspect of the present invention is a transient pharmaceuticalhemostatic liquid foam obtainable by a method according to the presentinvention, preferably for use as a hemostat, especially for thetreatment of wounds.

Another aspect of the present invention is a combination product,preferably a ready-to-use combination product, comprising apharmaceutical hemostatic liquid foam base preparation according to thepresent invention and a foaming gas.

Another aspect of the present invention is a kit for producing atransient pharmaceutical hemostatic liquid foam according to the presentinvention, comprising

(a) a first container comprising a pharmaceutical hemostatic liquid foambase preparation according to the present invention, and

(b) a second container containing a foaming gas.

Another aspect of the present invention is the use of a pharmaceuticalpreparation according to the present invention for the manufacture of ahemostat, especially a hemostat for the treatment of wounds.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure of skin (A), the view of created skin lesion(B) and application of flowable hemostatic foam according to the presentinvention (C).

FIG. 2 shows the degree of Bleeding Scale Scores (Bleeding score:

0 no bleeding—product saturated with blood

1 ooze—blood out of product but no blood drop

2 very mild—blood drop on the product

3 mild—blood drop streams down

4 moderate—small amount of blood streams down

5 severe—large amount of blood streams down.

FIG. 3 shows the product according to the present invention withthrombin 500 IU/ml+HSA 25% applied at 0 (A), 2 min (B), after irrigation(C) and at 5 min post application (D) in the porcine skin model.

FIG. 4 shows the product according to the present invention withthrombin 2500 IU/ml+HSA 25% applied at 0 (A), 2 min (B), afterirrigation (C) and at 5 min post application (D) in the porcine skinmodel.

FIG. 5 shows the product according to the present invention withthrombin 500 IU/ml+HSA 25% applied at 0 (A), 2 min (B), after irrigation(C) and at 10 min post application (D) in the liver abrasion model.

FIG. 6 shows the product according to the present invention withthrombin 2500 IU/ml+HSA 25% applied at 0 (A), 2 min (B), afterirrigation (C) and at 10 min post application (D) in the liver abrasionmodel.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a pharmaceutical hemostatic liquid foambase preparation comprising albumin as foaming agent and a fibrinogenprecipitating substance and optionally a coagulation inducing agent,wherein albumin as foaming agent is present in native form. With thefoam base according to the present invention, improved hemostatic foamsare provided which can be safely injected into tissue voids with lowvisibility and which are effective with no need for approximation. Noexcessive swelling or obstruction risks are obtained with the foamsaccording to the present invention. Moreover, the foam according to thepresent invention is specifically suitable in neuro/spine, laparoscopicand cardiovascular surgery.

It is important for the present invention that albumin is present in thefoam base in its native form, i.e. that albumin is properly folded andfully operative and functional. Accordingly, the foam base according tothe present invention is essentially free of denatured albumin.“Essentially free” means in this respect that the amount of denaturedalbumin should be 5% or lower, preferably 2% or lower, especially 1% orlower. Determination of the amount of denatured albumin can easily beperformed by methods available to a person skilled in the art, e.g. bynative gel electrophoresis or circular dichroism.

“Native form” as used in the present invention includes albumin whichhas undergone a pre-treatment, such as a pre-treatment by moderate heator a treatment for removing fatty acids or lipids, such as e.g.filtering over carbon filters.

Preferably, albumin is present in the pharmaceutical preparation asfoaming agent from 2 to 40%, preferably from 5 to 25% w/v, morepreferably from 10 to 20%, especially about 15%.

Preferably, the albumin used is an albumin which is pharmaceuticallyacceptable, especially human albumin from human blood plasma orrecombinant human protein. For certain uses (where this is appropriatewith respect to regulatory guidelines), also animal albumin may be used,e.g. plasmatic or recombinant bovine albumin or albumin (recombinant orplasmatic) from pig, goat, sheep, etc.

Preferred fibrinogen precipitating substances are fibrinogen cleavingsubstances, preferably selected from the group consisting of thrombin, asnake venom protease or a thrombin precursor. The most preferredfibrinogen cleaving substances is thrombin, which acts in convertingfibrinogen to fibrin, which assembles to a sealing, hemostatic fibrinnetwork.

Other preferred fibrinogen precipitating substances are selected fromthe group consisting of fibrinogen binding peptides, preferably GPRPcontaining peptides. Examples of such peptides are disclosed in WO2008/065388 A2. Those peptides may be bound to modified albumin. Suchpeptide constructs comprising modified albumin can be used as fibrinogenbinding substances according to the present invention; however, suchmodified albumin is not regarded as a foaming agent according to thepresent invention and would therefore not account to the foaming agentcomponent of the preparation according to the present invention.

The reason for this is that it is specifically preferred that albumin asfoaming agent is present in the foam base in a non-crosslinked form.Such albumin foaming agent therefore does neither contain residualamounts of crosslinking agents nor crosslinking moieties (from suchcrosslinking agent) between the albumin moieties.

Moreover, the foam base should lead to a transient foam to be applied toa wound. It is therefore preferred to omit any kind of chemicalstabilizers, such as any reactive substances, e.g. a crosslinking agent.It is specifically preferred to omit the addition of detergents to thepharmaceutical preparation according to the present invention.Accordingly the preparation is preferably essentially free ofdetergents. “Essentially free” means here a detergent concentrationwhich is 0.5% (v/v) or lower, preferably 0.1% or lower, especially 0.05%or lower.

The preferred fibrinogen precipitating substance according to thepresent invention is thrombin. Thrombin is preferably present in aconcentration of 0.1 IU/ml to 10000 IU/ml, more preferred of 1 IU/ml to8000 IU/ml, especially 100 to 5000 IU/ml.

It is also preferred to add “active substances”, preferably assolutions, but also solids are possible, to the pharmaceuticalhemostatic liquid foam base preparation according to the presentinvention to make the foams even more effective and useful as hemostaticagents. Advantages of presenting such active substances in a foamformulation rather than in solution include the high surface area forenhanced contact between blood and the active substance, and the factthat the active substance is in contact with the bleeding tissue for anextended amount of time compared to a solution, which tends to run offfrom the wound if not perfectly level.

These active substances can be classified by their mode of action inenhancing the hemostatic activity of the pharmaceutical hemostaticliquid foam base preparation according to the present invention.

Coagulation Factors: These typically are enzymes found in thecoagulation cascade and act by enhancing the coagulation cascade of thepatient. Examples of coagulation factors, which can be used either aloneor in combination are FVII, FVIII, FIX, FX, FXIII, their activatedforms, such as e.g. factor VIIa, or FEIBA.

Vitamin K (K₁ and K₃) can be also considered as coagulant to be added toa foam formulation as it helps to regulate the activity of a number ofproteins involved in platelet aggregation, or blood clotting. Vitamin Kcan help to activate factor X, IX, VII and prothrombin, all proteinsthat promote blood clot formation.

Platelet activators: Activation of platelets is an essential step inachieving hemostasis, therefore the addition of platelet activators canbe a powerful way of enhancing the hemostatic potency of such foamformulations. Collagen is known as a platelet activator, as isthromboxane A2, or arachidonic acid, (the precursor of thromboxane A2),ADP (adenosine diphosphate) and many other substances. In a preferredembodiment collagen is added to the composition of the presentinvention, especially preferred collagen in solid form is present.

Coagulation inducing substances: are agents that induce bloodcoagulation (thrombogenesis), i.e. the process by which blood formsclots. Examples of such agents are agents that are naturally inducingcoagulation, such as tissue factor, activated platelets, factors IIa(thrombin), Va, VIIa, VIIa, IXa, Xa, XIa, XIIa, XIIIa; orantihemorrhagic agents such as zeolithes, anhydrous aluminium sulfate,potassium alum, titanium dioxide, (microfibrillar) collagen, chitosan,etc.; collagen and ((water) soluble and insoluble) chitosan beingspecifically preferred coagulation inducing substances. Coagulationinducing substances such as chitosan can be added to further inducecoagulation and support the hemostatic characteristics of thepreparation according to the present invention. In a preferredembodiment chitosan is present in the composition of the presentinvention either in solid form or as a solution.

Vasocontrictors: Vasoconstrictors are molecules that lead to a transientnarrowing of blood vessel luminal diameter—as such they are useful inhemostasis by temporarily reducing blood flow rate, primarily in thevicinity of the applied foam.

According to another aspect, the present invention relates to a methodfor preparing a pharmaceutical hemostatic liquid foam, wherein apharmaceutical hemostatic liquid foam base preparation according to thepresent invention is contacted with a foaming gas so as to obtain apharmaceutical hemostatic liquid foam.

Preferably, the foaming gas is selected from the group consisting ofair, N₂, O₂, N₂O, CO₂, propane, butane, dimethylether, or partlyfluorinated hydrocarbons (HFCs), especially 1,1,1,2-Tetrafluoroethane(HFC134a) or 1,1,1,2,3,3,3-Heptafluoropropane (HFC227ea). The foaminggas is preferably used in an amount of 1 to 30% (w/v), especially 5 to15% (w/v).

The foaming gas may be provided under pressure. For example, thepressurized gas may be introduced into the pharmaceutical hemostaticliquid foam base preparation by a nozzle. It is e.g. possible tointroduce the foaming gas directly from a gas cylinder containing thepressurized gas to the foam base.

Accordingly, the foam is preferably created by contacting thepharmaceutical hemostatic liquid foam base preparation with the foaminggas by applying pressure. The pressure may e.g. be implied by apressurised foaming gas container or by applying physical force by the(hand of the) user of the present foam base, e.g. the surgeon or surgeonassistants.

The mixing of the foaming gas could also be performed under pressure inan appropriate aerosol containing container wherein the foaming gas suchas a propellant like R134a, R227ea, DME etc., but not gases such as air,N₂, CO₂, butane and propane and the like, is in a liquid state and assuch solubilised in the liquid formulation of the fibrinogenprecipitating substance and/or dispersed therein;

In a preferred embodiment of the method, the pharmaceutical hemostaticliquid foam base preparation is provided in a first container and thefoaming gas is provided in a second container. Then the first and thesecond container are connected so as to allow the pharmaceuticalhemostatic liquid foam base preparation to be mixed with the foaming gasto obtain a foam. Preferably, the first and the second container isconnected with a mixing device, especially a mixing device selected fromthe group consisting of a filter material, a porous disc device, athree-dimensional lattice or a matrix material. Preferred examples ofsuch mixing devices (and suitable containers therefor) are disclosede.g. in WO 2007/084919 A1, WO 2009/120432 A2 or WO 2009/120433 A2.

In a further preferred embodiment mixing can be done in using specificcontainers or aerosol cans. All components of the formulation are addedin solution to a pressure-proof container, which is then closed using avalve assembly of some sort. The propellant gas or liquefied gas (as inthe case of R134a) is then added, either through the valve itself orthrough a secondary port. The propellant either dissolves completely inthe liquid formulation, dissolves partially, or the propellant forms aseparate phase in the container. In any case, upon dispensing theproduct through the valve, the gas expands or the liquid gas evaporatesfrom within the liquid formulation, thereby forming a foam upon release.

According to another preferred embodiment of the method, thepharmaceutical hemostatic liquid foam base preparation and the foaminggas is provided in a container and the foam is obtained by mixing thepharmaceutical hemostatic liquid foam base preparation with the foaminggas.

Another aspect of the present invention relates to a combinationproduct, preferably a ready-to-use combination product, comprising apharmaceutical hemostatic liquid foam base preparation according to thepresent invention and a foaming gas, preferably a foaming gas selectedfrom the group consisting of air, N₂, O₂, N₂O, CO₂, propane, butane,dimethylether, or partly fluorinated hydrocarbons (HFCs), especially1,1,1,2-Tetrafluoroethane (HFC134a) or 1,1,1,2,3,3,3-Heptafluoropropane(HFC227ea). With such a combination product the foam can easily beproduced (e.g. by mixing (especially swooshing) the foam base with thefoaming gas in the (common) container of the combination product). Thiswould allow the production of the foam from one container (e.g. a vialor a syringe) immediately at the place of surgery.

According to another aspect, the present invention relates to atransient pharmaceutical hemostatic liquid foam obtainable by a methodaccording to the present invention, preferably for use as a hemostat,especially for the treatment of wounds.

The foam according to the present invention is not stable so that itdegrades shortly after administration e.g. to a wound. “Transient”according to the present invention therefore has the meaning that thefoam is chemically unstabilized, essentially free of any reactivesubstances, such as e.g. free of crosslinking agents, and degradesshortly after hemostasis has been achieved, i.e. up to about 5 times thetime necessary to achieve hemostasis, e.g. about 10 minutes.

According to another aspect, the present invention relates to a kit forproducing a transient pharmaceutical hemostatic liquid foam according tothe present invention, the kit comprising

(a) a first container comprising a pharmaceutical hemostatic liquid foambase preparation according to the present invention, and

(b) a second container containing a foaming gas.

The foaming gas is preferably selected from the group consisting of air,N₂, O₂, N₂O, CO₂, propane, butane, dimethylether, or partly fluorinatedhydrocarbons (HFCs), especially 1,1,1,2-Tetrafluoroethane (HFC134a) or1,1,1,2,3,3,3-Heptafluoropropane (HFC227ea).

In a preferred embodiment, the first and the second container areconnectable by a mixing device, especially a mixing device selected fromthe group consisting of a filter material, a porous disc device, athree-dimensional lattice or a matrix material. In a further preferredembodiment it is also possible to produce a composition of the presentinvention without using a mixing device.

Preferably, the kit is provided in a form, wherein the containers aresyringe containers.

Also preferred is the use of aerosol cans for producing a composition ofthe present invention.

According to another aspect, the present invention relates to the use ofa pharmaceutical preparation according to the present invention for themanufacture of a hemostat, especially a hemostat for the treatment ofwounds.

The foam base preparation according to the present invention containinghuman serum albumin (HSA) in an aqueous solution can be expanded inseveral different directions. Stable, fine-pored foams can be producedusing the pharmaceutical hemostatic liquid foam base preparationaccording to the present invention with a preferred concentration of 5to 25% albumin (w/v fluid phase). The aqueous solution can bephysiological saline (0.9% (w/v), phosphate buffered saline, preferablya solution containing up to 10% (w/v fluid phase) sodium chloride,preferably a solution containing CaCl₂ in concentrations up to 40 mM. Itis also preferred to use preparations containing sucrose inconcentrations of up to 2.5% (w/v fluid phase). In another embodiment itis preferred to use collagen, such as collagen in solid form and/orsoluble or insoluble chitosan. A preferred composition of the presentinvention includes thrombin, albumin and collagen and/or (in)solublechitosan.

For the example section, foams were created from these preparations e.g.by mixing the solutions in a ratio of typically 1:5 with air (1 mlfluid+4 ml of air). Other fluid to gas ratios are also possible, thelimits of the ratio can easily be established for each individualpreparation and the intended use and consistency of the foam.Accordingly, ratios ranging from 1:10 to 10:1 (air:fluid) are wellwithin the scope of this invention. Mixing can e.g. be achieved byfilling one 5 ml male luer syringe (e.g. B. Braun Injekt) with 1 ml ofthe respective solution, and another such syringe with 4 ml of air.These two syringes were connected by means for a female-female luerconnector. The system was foamed by repeated transfer back and forth ofthe syringe contents until the entire volume was filled with afine-pored foam (typically a total of 20 passes). Foaming is improved ifthe female-female luer contains a porous polypropylene disk according toWO 2007/084919 A1.

Other preferred methods of mixing are:

-   -   In-line foam dispenser: Using a gas driven foam set that        consisted of a Tisseel Spray head coupled with a female-male        luer connector with 2 porous polypropylene disk according to WO        2007/084919 A1 (e.g. FIG. 34). Flowing air through the assembly        with 1 bar above atmosphere, and then dispensing the        pharmaceutical hemostatic liquid foam base preparation according        to the present invention into the air flow and through the 2        mixing disks yielded a stable HSA foam without the need of        mechanical mixing.    -   Introducing the pharmaceutical hemostatic liquid foam base        preparation according to the present invention into a        pressure-stable vessel, adding N₂O to the solution in the        pressure-stable vessel, shaking the solution in the        N₂O-pressurized vessel, and then releasing the solution to        atmospheric pressure through an applicator. In this process,        analogous to the formation of whipped cream in the same device        and using the same method, a foam was formed. The foam, however,        was not as stable as the one created by mechanical mixing with        air.    -   A foam base with thrombin may also be contacted with R134a or        propane/butane, which upon release forms a stable transient foam        according to the present invention. Coagulation inducing agents        such as e.g. collagen and/or (in)soluble chitosan as mentioned        above may be added.

Based on the pharmaceutical hemostatic liquid foam base preparationaccording to the present invention containing HSA in aqueous solutions,formulations containing thrombin in concentrations up to 2500 IU/ml weremade and tested in the example section.

The intended use of the pharmaceutical hemostatic liquid foam basepreparation according to the present invention is to provide a(transient) hemostatic foam which is e.g. specifically suitable for thetreatment of surgical or emergency bleeds on tissue surfaces or incavities where the application of conventional topical hemostats isdifficult or impractical. The foam according to the present invention isintended to achieve hemostasis without the need for approximation of thebleeding site, which make the invention suitable for use in neurologicalindications where little or no pressure can be applied to the tissues.This also allows the application of the foam according to the presentinvention in bleeding situations where the exact location of the bleedcannot be determined visually, either because the overall visibility ofthe surgical field is difficult to achieve (cavities, diffuse bleedingsin laparoscopic surgery, etc.) or where achieving hemostasis quickly iscritical for the survival of the patient. Hemostasis can be achievedeither by mechanically sealing the bleeding surfaces, thus activatingthe blood clotting cascade inside the capillaries where blood flow isimpeded, or by activating blood clotting by chemical, biochemical orphysical means, using agents that are delivered within the foamformulations according to the present invention. If the primary mode ofaction of the foam formulation is by sealing, other sealing indicationscan also be addressed by these formulations.

The foam formulation according to the present invention can also be seenas a means to achieve extended contact between a bleeding surface and astand-alone thrombin product frequently used in some geographies as ameans to achieve hemostasis.

EXAMPLES Example 1 1. Summary

Two different hemostats in the form of a “flowable foam” according tothe present invention (the pharmaceutical hemostatic liquid foam basepreparation according to the present invention) have been tested in non-and heparinized pig in partial thickness skin lesion and in liverabrasion model.

Flowable foams according to the present invention were prepared with twodifferent initial thrombin concentrations of 500 IU/ml and 2500 IU/ml.In both formulations 25% solution of human serum albumin (HSA) was usedas a foaming agent.

Hemostatic efficacy of all formulations was initially tested in partialthickness porcine skin lesion, in non-heparinized animal. The lesionswith diameter of 2×3 mm and thickness of 1 mm were created usingdermatome starting from rump and continued to back. In this type oflesion only diffuse, weak bleeding was achieved. The samples tested wereapplied 1 min after lesion was created and irrigation of the product wasperformed 2 min post application. There was no product approximation.

All formulations tested showed satisfactory hemostatic efficacy in theporcine skin model. An overall perception was that the formulation withhigher concentration of thrombin (2500 IU/ml) was slightly better thatformulation with lower Thrombin concentration.

In the second model—swine liver abrasion model when the pig was stillnot heparinized there was no perceptible differences in hemostaticperformance of the products.

At the end of in vivo test, the formulation with higher thrombinconcentration was tested in liver abrasion model in heparinized pig andat this condition the samples could not stop bleeding.

In the case of strong bleeding in heparinized animal flowable foamhemostats require further optimization as under such conditions proposedformulations were not effective.

2. Scope of the Experiments

A hemostatic, adherent foam formulation that is biocompatible and fullybio-resorbable is provided with the present invention as one of anappealing option for new generation product in hemostasis.

The foam should safely be injected into tissue voids with low visibilityand should be effective with no need for approximation. Additionalimportant requirement of the foam hemostat is absence of excessiveswelling or obstruction risk. Main application of such flowablehemostatic foam involves neuro/spine, laparoscopic and cardiovascularsurgery.

Flowable foam systems according to the present invention based on humanserum albumin mixed with thrombin have been designed. It has been foundthat concentrated solution of HSA when mixed 1:3 with air, results withstable over longer time (10 min) foam that can be prepared using twosyringes connected with special mixing unit. Such foam can be furtherapplied through the same application tip that is used in FlosealHemostatic Matrix.

The purpose of these experiments was to test hemostatic efficacy ofselected flowable foam formulations according to the present invention.The formulations were tested at moderate bleeding in two differentmodels in non-heparinized pig. In the first model the formulations weretested in partial thickness skin lesion and in the second model in liverabrasion lesion. Efficacy of the samples were assessed onlyqualitatively.

3. Materials

3.1 Used Raw Materials

Material Supplier Lot Number Thrombin SD TIM5 500 IU/ml US 5 ml BaxterVNF4K001A3.2 SolutionsHSA 25%Calcium-chloride sol. 40 mM 5 mlThrombin sol. 500 IU/ml in CaCl₂ 40 mMThrombin solution 2500 IU/ml in CaCl₂ 40 mMSodium-chloride solution 0.9%3.3 Equipment/Disposables

Equipment/disposables Supplier Syringes 5 ml Luer-Lock solo BBraunRapid-fill-connector, Luer-lock-to-luer-lock Baxa Mixing device “Mix-F”Baxter FloSeal applicators Tip Large 6.35 mm Baxter Abrasion device, d =18 mm Dremel Timer Huger

4. Methods

4.1 Sample Preparation

4.1.1 Thrombin 500 IU/ml+HSA 25%+4 ml Air

Thrombin 500 IU/ml in CaCl₂ 40 mM+4 ml Air:

Thrombin 500 IU/ml lyo vial was dissolved in 5 ml calcium-chloridesolution 40 mM. 0.5 ml of the Thrombin solution and 4 ml air were drawnup in 5 ml syringes each. The syringes were then closed with caps andstored at −20° C. until further use.

HSA 25%:

0.5 ml of human albumin serum 25% was drawn up in 5 ml syringes each.The syringes were closed with caps and stored at +4° C. until furtheruse.

4.1.2 Thrombin 2500 IU/ml+HSA 25%+4 ml Air

Thrombin 2500 IU/ml in CaCl₂ 40 mM+4 ml Air:

Thrombin 500 IU/ml lyo vials were dissolved in 1 ml calcium-chloridesolution 40 mM each. 0.5 ml of the Thrombin solution and 4 ml air weredrawn up in 5 ml syringes each. The syringes were then closed with capsand stored at −20° C. until further use.

HSA 25%:

0.5 ml of human albumin serum 25% was drawn up in 5 ml syringes each.The syringes were closed with caps and stored at +4° C. until furtheruse.

4.2 Sample Reconstitution

4.2.1 Thrombin 500 IU/ml+HSA 25%:

0.5 ml Thrombin 500 IU/ml in CaCl₂ 40 mM

+

0.5 ml HSA 25%

↓+“Mix-F”

Swooshing 10× (20 passes)

↓

Application with applicator tip 6.35 cm

4.2.2 Thrombin 2500 IU/ml+HSA 25%:

0.5 ml Thrombin 2500 IU/ml in CaCl₂ 40 mM

+

0.5 ml HSA 25%

↓+“Mix-F”

Swooshing 10× (20 passes)

↓

Application with applicator tip 6.35 cm

4.3 Surgical Procedure and Bleeding Assessment

Initially, all foam formulations were tested in porcine skin lesion andliver abrasion model in non-heparinized animal. Only one sample fromformulation with higher thrombin concentration (2500 UI/ml) was testedin liver abrasion model in heparinized pig.

4.3.1 1 mm Partial Thickness Porcine Skin Lesion

The lesions with diameter of about 2×3 mm and thickness of 1 mm werecreated using dermatome starting from rump and continued to back ofnon-heparinized pig. After lesion was created, exact size of the skindefect was measured. A product was applied 1 min after lesion wascreated. Before application the excess of blood from the lesion wasswabbed (blotted) with gauze and the product was homogenouslydistributed on the lesion as illustrated in FIGS. 1 (B) and (C). In thistype of lesion only diffuse, weak bleeding was achieved. The samplestested were applied without approximation. Irrigation of the product wasperformed 2 and 5 min post application.

4.3.2 Swine Liver Abrasion Model

In this model, liver abrasions were created using a hand-drill dremelfixed with medium grade sandpaper. The refined model reduces variabilityby using standardized 1.8 cm diameter, about 1 mm deep lesions. With aflat, round, rotating abrasion tool a circular bleeding wound (1.8 cmdiameter) was created on the liver lobe surface of a pig. Beforeapplication the excess of blood from the lesion was swabbed (blotted)with gauze and next product was homogenously distributed on the lesion.There was no product approximation. The hemostatic efficacy wasevaluated after 2, 3, 5 and 10 minutes. Excess of product was irrigatedwith Ringer solution at 5 min post application.

4.3.3 Bleeding Assessment

Bleeding from the crated lesion in both models (skin and liver) wasassessed qualitatively (subjectively) according to the scale presentedin FIG. 2. Product was prepared and applied onto created lesion. Therewas no approximation after product has been applied. Excess of productwas irrigated with Ringer solution in 20 ml syringe. The degree ofbleeding was assessed after 30 sec, and then approximately after 1, 2, 5and 10 min. Product saturated with blood but without active bleedingwere scored as 0. The same saline solution was used to irrigate excessproduct from the lesion after the 5 minute since application.

Bleeding Score:

0 no bleeding product saturated with blood 1 ooze blood out of productbut no blood drop 2 very mild blood drop on the product 3 mild blooddrop streams down 4 moderate small amount of blood streams down 5 severelarge amount of blood streams down

5. Results

Each tested product was in the form of (transient) flowable foamaccording to the present invention.

5.1 Thrombin 500 IU/ml+HSA 25%; Porcine Skin Model

The results are depicted in FIG. 3. The foam was prepared and applied toa bleeding lesion. With time liquid oozing was observed most probablydue to gravitation of liquid (drainage of liquid to the foam base).Excess of the product could be irrigated away with Ringer solution. Theproduct was tested in three applications with final score “0”—hemostaticsuccess.

5.2. Thrombin 2500 IU/ml+HSA 25%; Porcine Skin Model

The results are depicted in FIG. 4. The foam was prepared and applied toa bleeding lesion. Excess of the product could be irrigated away withRinger solution. The product was tested in three applications with finalscore “0”—hemostatic success.

5.3 Thrombin 500 IU/ml+HSA 25%; Liver Abrasion Model

The results are depicted in FIG. 5. Two samples were tested with finalscore “0”—hemostatic success.

5.4 Thrombin 2500 IU/ml+HSA 25%; Liver Abrasion Model

The results are depicted in FIG. 6. Two samples were tested with finalscore “0”—hemostatic success.

6. Discussion

In order to obtain the liquid foam according to the present inventionwas mixed 1:3 with air using two syringes connected with special mixingunit. Such foams were observed to be stable over 10 min and could befurther applied through an application tip the same as it is used inFloseal Hemostatic Matrix.

The purpose of these experiments was to test hemostatic efficacy ofthese foam formulations. The formulations were tested at moderatebleeding in two different models in non-heparinized pig. In the firstmodel the formulations were tested in partial thickness skin lesion andin the second model in liver abrasion lesion. Efficacy of the sampleswas assessed only qualitatively.

Flowable foams according to the present invention were prepared with twodifferent initial thrombin concentrations of 500 IU/ml and 2500 IU/ml.In both formulations 25% solution of HSA was used as a foaming agent.

Hemostatic efficacy of all formulations was initially tested in partialthickness porcine skin lesion, in non-heparinized animal. The lesionswith diameter of 2×3 mm and thickness of 1 mm were created usingdermatome starting from rump and continued to back. In this type oflesion only diffuse, weak bleeding was achieved. The samples tested wereapplied 1 min after the lesion was created and irrigation of the productwas performed 2 min post application. There was no productapproximation.

All formulations tested showed satisfactory hemostatic efficacy in theporcine skin model. An overall perception was that the formulation withhigher concentration of thrombin (2500 IU/ml) was slightly better thatformulation with lower thrombin concentration.

In the second model—swine liver abrasion model when the pig was stillnot heparinized there was no perceptible differences in hemostaticperformance of the products.

Example 2

A Hemostatic Foam based on human serum albumin (HSA), thrombin and airwas prepared by mixing one part of 25% HSA solution with one part of athrombin solution containing 500 IU/ml thrombin and 5% HSA in a syringewith at least 5 ml volume. The third component was air at 80 vol %; theair was in a second syringe of the same size. The foam was prepared byrapidly transferring the contents of both syringes back and forth atleast 20 times through a connector fitted with a sintered porouspolypropylene disk (called “Mix F”). This produced a stable, whitetransient foam with excellent hemostatic properties.

Example 3

A foam prepared as in example 2 was applied to a liver abrasion wound ofa pig heparinized to 1.5-2×ACT, which presents mild to moderate bleeds.No breakthrough bleeding was observed in the first 5 minutes afterapplication. After 5 minutes, the remaining foam was irrigated withsterile saline. A stable foam blood clot had formed that presented amechanically stable, yet compliant and thin wound cover.

Example 4

Further foam stabilization over the formulation presented in example 2can be achieved as follows: HSA 25%-thrombin (5% HSA, 500 IU/ml) 1:1mixture containing additionally with 5% (w/v) of polyvinylpyrolidone(PVP). 0.5 g of PVP were dissolved in 10 ml mixture of 25% HSA andthrombin 500 IU/ml. From this solution 1 ml was drawn into a 5 mlsyringe and mixed with 4 ml of air in a second 5 ml syringe by rapidlytransferring the contents of both syringes back and forth at least 20times through a connector fitted with a sintered porous polypropylenedisk (called “Mix F”). The final formulation contains: 12.5% HSA, 250IU/ml thrombin and 5% PVP.

Example 5

When placing the foams produced according to example 2 and 4 onto apetri dish which contained 5 ml of human plasma and monitoring thediameter of the foam over the time course of 20 minutes (foam stabilityassay), a significantly slower spreading of the foam produced accordingto example 3 was observed, hence the foam was more stable.

Example 6

Chitosan 0.5% foam: From a 1% solution of chitosan, 0.5 ml were added to1 ml of the 25% HSA and thrombin solution of example 2. The solution in5 ml syringe was mixed with 4 ml of air in the second 5 ml syringethrough Mix F connector to produce a stable foam. When subjected to thefoam stability test as described in example 5, and significantly slowerspreading of the foam produced according to example 6 was observed,hence the foam was more stable.

Example 7

Chitosan 0.5% foam in aerosol spray can: From a 1% solution of chitosan,0.5 ml was added to 1 ml of the 25% HSA and thrombin solution of example2. 10 ml of this solution were filled in a 50 ml aerosol can, and avalve assembly was fitted. 2 g of R134a were filled into the aerosol canthrough the valve assembly. Upon dispensing of the liquid, a stable foamwas formed with the same characteristics as the foam produced manuallyin syringes.

Example 8

A aerosol spray can according to example 7 was used to dispense athrombin containing liquid foam onto an abrasion lesion of the pleura ina porcine animal model. The foam was easy to place on the verticalpleural wall, and remained in place until hemostasis was achieved. Ifnecessary, the product can be reapplied to extend the contact time.

Example 9

Foam formulation with solid collagen powder: 34 mg crosslinked collagenwere mixed with 1 ml 25% HSA and 1 ml of thrombin 1500 IU/ml solution.To this suspension 150 μl of 1% chitosan solution were added and mixedby rapidly transferring the contents of both syringes back and forth atleast 20 times between two 10 ml B. Braun syringes. The amount of theair was set to 4 ml. The foam was produced again by the same process ofrapidly transferring the contents of both syringes back and forth atleast 20 times. A stable, white transient foam was produced that showedbetter efficacy in the porcine animal model described in example 3.

Example 10

Collagen (100 mg) foam: 1.5 ml 25 wt % HSA was mixed by swooshing with100 mg of milled collagen paste and 1 ml of air. The amount of air inthe foam was incrementally (each time 1 ml) increased up to total amountof 3 ml in order to obtain foam like structure. Preparation of thisformulation was done using 10 ml syringes.

The invention claimed is:
 1. A combination product comprising: apharmaceutical hemostatic liquid foam base preparation, wherein thepharmaceutical liquid foam base preparation comprises albumin as foamingagent and a fibrinogen precipitating substance, wherein the albumin asfoaming agent is present in native form, and a foaming gas, wherein: thepharmaceutical hemostatic liquid foam base preparation is a solution andthe concentration of the albumin in the pharmaceutical hemostatic liquidfoam base preparation is from 5% to 40% w/v.
 2. A pharmaceuticalhemostatic liquid foam obtainable by a method comprising: providing apharmaceutical hemostatic liquid foam base preparation comprisingalbumin and thrombin, wherein: albumin is present in its native form,albumin is not crosslinked, and thrombin is present in a concentrationof 100 to 10000 IU/ml; and contacting the pharmaceutical hemostaticliquid foam base preparation with a foaming gas so as to obtain thepharmaceutical hemostatic liquid foam, wherein: the pharmaceuticalhemostatic liquid foam base preparation is a solution, thepharmaceutical hemostatic liquid foam degrades in less than or equal toabout 5 times the time necessary to achieve hemostasis, and theconcentration of the albumin in the pharmaceutical hemostatic liquidfoam base preparation is from 5% to 40% w/v.
 3. A kit for producing apharmaceutical hemostatic liquid foam, comprising (a) a first containercomprising a pharmaceutical hemostatic liquid foam base preparation,wherein the pharmaceutical hemostatic liquid foam base preparationcomprises: albumin in its native form, and a fibrinogen precipitatingsubstance, and (b) a second container containing a foaming gas, wherein:the pharmaceutical hemostatic liquid foam base preparation is a solutionand the concentration of the albumin in the pharmaceutical hemostaticliquid foam base preparation is from 5% to 40% w/v.
 4. The kit accordingto claim 3, wherein the foaming gas is selected from the groupconsisting of air, N₂, O₂, N₂O, CO₂, propane, butane, dimethylether, orpartly fluorinated hydrocarbons (HFCs).
 5. The kit according to claim 3,wherein the first and the second container are connectable by a mixingdevice.
 6. The kit according to claim 3, wherein the containers aresyringe containers.
 7. The kit according to claim 3, wherein theconcentration of the albumin in the pharmaceutical hemostatic liquidfoam base preparation is from 10% to 20% w/v.
 8. The kit according toclaim 3, wherein the concentration of the albumin in the pharmaceuticalhemostatic liquid foam base preparation is about 15% w/v.
 9. The kitaccording to claim 3, wherein the pharmaceutical hemostatic liquid foambase preparation is essentially free of detergents.
 10. The kitaccording to claim 3, wherein the pharmaceutical hemostatic liquid foambase preparation further comprises a coagulation inducing agent.
 11. Thekit according to claim 3, wherein: the pharmaceutical liquid foam basepreparation further comprises denatured albumin, and 5% or lower of thecombined albumin in its native form and denatured albumin in thepharmaceutical hemostatic liquid foam base preparation is denaturedalbumin.
 12. The kit according to claim 3, wherein: the pharmaceuticalliquid foam base preparation further comprises denatured albumin, and 2%or lower of the combined albumin in its native form and denaturedalbumin in the pharmaceutical hemostatic liquid foam base preparation isdenatured.
 13. The kit according to claim 3, wherein: the pharmaceuticalliquid foam base preparation further comprises denatured albumin, and 1%or lower of the combined albumin in its native form and denaturedalbumin in the pharmaceutical hemostatic liquid foam base preparation isdenatured.
 14. The kit according to claim 3, wherein the pharmaceuticalhemostatic liquid foam base preparation is free of a crosslinking agent.15. The combination product according to claim 1, wherein the foaminggas is selected from the group consisting of consisting of air, N₂, O₂,N₂O, CO₂, or partly fluorinated hydrocarbons (HFCs).
 16. Thepharmaceutical hemostatic liquid foam according to claim 2, wherein thepharmaceutical hemostatic liquid foam is stable for less than or equalto 10 minutes.
 17. The kit according to claim 3, wherein thepharmaceutical hemostatic liquid foam base preparation consists ofalbumin in its native form, the fibrinogen precipitating substance,saline, and at least one of a coagulation factor, a platelet activator,a coagulation inducing substance, or a vasoconstrictor.
 18. Thepharmaceutical hemostatic liquid foam according to claim 2, wherein thepharmaceutical hemostatic liquid foam consists of the foaming gas andthe pharmaceutical hemostatic foam base preparation.
 19. The kitaccording to claim 3, wherein the contents of the second containerconsist of air.
 20. The kit according to claim 3, further comprising amixing device wherein the first and second containers are connectable bythe mixing device.